Device

Part:BBa_K4813025

Designed by: Lam Chung Hin   Group: iGEM23_HongKong-JSS   (2023-10-08)


Reverse HxlR-K13A-dTomato formaldehyde sensing chromoprotein reporter

This composite component comprises several elements, including a HxlR protein with a K13A mutation (BBa_K4813009), a constitutive strong promoter (BBa_J23100), wild-type BRH1&2 binding sites for HxlR protein (BBa_K4813016), a wild-type promoter for Bacillus subtilis HxlAB operon (BBa_K4813017), a wild-type Bacillus subtilis RBS site (BBa_K4813021), an E. coli codon-optimized chromoprotein and red fluorescent protein dTomato coding sequence (BBa_K4813000), and a strong double terminator (BBa_B0015). Additionally, the 5' and 3' ends of the composite part feature a 20 base pair overlap sequence designed for the pUC19 EcoRI restriction site for NEBuilder HiFi assembly ((BBa_K4813007) & (BBa_K4813008)).


Construct map of K4813025
Fig. 1 Construct map showing the basic parts in this composite part.


The HxlR protein is a formaldehyde-responsive transcription factor from Bacillus subtilis [1,2]. In the presence of formaldehyde, wild-type HxlR protein binds to two specific binding sites (BRH1&2) and activates the expression of the downstream hxlAB operon in Bacillus subtilis [2]. According to literature, when the 13th amino acid in HxlR protein, Lysine, was replaced by Alanine, mutated HxlR-K13A protein showed a more optimal conformation for DNA binding and transcription activation than the wild-type HxlR protein [1].

In this composite component, we incorporated the mutated K13A version of HxlR gene to examine if HxlR-K13A can lead to an enhanced expression of a red-colored dTomato chromoprotein when formaldehyde is present, comparing to our another composite component expressing wild-type HxlR (BBa_K4813014). In our project, this composite component serves the purpose of detecting the presence of formaldehyde by inducing a color change in the host E. coli.


Usage and Biology

Validation of clones

We obtained the part constructs in the form of gBlocks from IDT. We then performed HiFi assembly (NEB) to assemble these constructs in pUC19 vectors. To verify the clones, we ordered pUC19 M13 PCR primers and performed colony PCR on the resulting transformants. Initially, we faced several unsuccessful attempts at cloning. However, after multiple trials, we successfully cloned the desired construct and transformed it into E. coli.


Colony PCR result of K4813009
Fig. 2 The gel result of colony PCR for verifying the clones of this part. Lane 1 is the DNA ladder (1kbp+ NEB), Lane 2-7 are the colonies, showing the expected band size of ~1.5kbp. Lane 9 is the no template control.


Figure 2 shows the gel electrophoresis result of a successful cloning attempt from colony PCR, showing the presence of a band of the predicted size (~1.5kbp) corresponding to the M13 amplification of our inserted constructs. It is worth mentioning that during our observations, we noticed that some colonies on the plate showed a pink colour, indicating potential issues with leaky expression. Therefore, we decided to select a white-colored colony for further investigation and proceed with our functional assay involving formaldehyde.


Functional assay with formaldehyde

The E. coli strain expressing the construct was then cultured in 10 mL of LB broth with ampicillin for a duration of 12 hours. During this period, formaldehyde was added in concentrations of 500 uM and 1000 uM. Following the incubation, the culture was harvested and subjected to centrifugation, resulting in the formation of a pellet for easier observation of any color changes.

In this modified strain, we noticed that there was no change in color in the pellets of the engineered E. coli bacteria. However, our other formaldehyde sensing device showed a positive result with a noticeable color change. If you're interested in learning more about this comparison and the specific details, please refer to the section on (BBa_K4813002) for further information.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 384
    Illegal NheI site found at 407
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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